The present invention relates generally to the storage of blood and blood components, and more specifically, to an improved system for adding a cryopreservation solution to red blood cells prior to freezing and for washing that solution from the red blood cells prior to their use.
Human blood predominantly includes three types of specialized cells: red blood cells, white blood cells, and platelets. These cells are suspended in a complex aqueous solution of proteins and other chemicals called plasma. Although in the past blood transfusions have used whole blood, the current trend is to collect and transfuse only those blood components required by a particular patient. This approach preserves the available blood supply and in many cases is better for the patient, since the patient is not exposed to unnecessary blood components.
Blood components are typically obtained from a donor following whole blood collection. A disposable blood collection set, including a harness (e.g., tubing and connectors), a phlebotomy needle and one or more collection bags, is utilized to collect the whole blood from the donor. In particular, the phlebotomy needle is inserted into the donor""s arm and blood flows under gravity into a collection bag which may contain an anticoagulant. Thereafter, the whole blood may be provided to a blood-processing machine for separation into one or more desired components. The machine includes a rotatable separation chamber which subjects the whole blood to many times the force of gravity, thereby separating the various blood components according to their densities. That is, the more dense components, such as red blood cells (RBCs), accumulate within the chamber at its outer periphery, while the less dense components are withdrawn through an outlet port. When the separation process is complete, the RBCs remaining in the separation chamber are removed. Blood components may also be obtained through apheresis in which whole blood from the donor is directly provided to the blood processing machine for separation and collection. With this method, any uncollected blood components may be returned directly to the donor.
The collected blood components may then be stored before transfusion back to the donor or to some other patient. For example, the individual blood components, such as RBCs, may be refrigerated at approximately 4xc2x0 C. for several days. Procedures are also known for freezing RBCs to further extend their shelf-life. For example, RBCs may be stored at approximately xe2x88x9280xc2x0 C. or xe2x88x92160xc2x0 C., depending on how they are prepared, for several years.
In particular, RBCs are typically preserved with glycerol, prior to freezing, which crosses the cell membranes. Without glycerol, the RBCs would not survive the freezing process. U.S. Pat. Reissue No. 33,924 to Valeri and entitled Apparatus and Method for Storing and Processing Blood discloses a method and apparatus for glycerolizing RBCs prior to freezing and also for washing the glycerol from thawed RBCs prior to transfusion. In particular, a three step process is used to introduce glycerol into a bag containing RBCs. First, the RBC bag is placed on a shaker platform operating at approximately 180 oscillations per minute, and 50 ml of a glycerol solution is gravity fed into the bag. The shaker platform is then turned off for about five minutes, allowing the RBCs and glycerol solution to equilibrate. Next, the shaker platform is turned back on and a second volume of approximately 50 ml of glycerol solution is added to the bag. Again, the shaker platform is turned off and the RBCs and glycerol solution are allowed to equilibrate for approximately two minutes. The bag is then removed from the shaker platform and a third volume of approximately 400 ml of glycerol solution is added while an operator applies manual agitation to the bag.
The bag is then loaded onto a bag centrifuge device to concentrate the glycerolized RBCs. This results in the bag containing concentrated-glycerolized RBCs and a supernatant glycerol solution. To remove the supernatant, the bag is placed m a plasma extractor. The bag may then be sealed in an overwrap bag and placed in a freezer operating at xe2x88x9280xc2x0 C. As noted in the ""924 patent, the entire process must be completed within four hours and results in glycerolized RBCs having a hematocrit of approximately 60%.
Thus, the prior art glycerolization process is a time-consuming and labor intensive task. It also requires a highly skilled operator to ensure that the glycerol solution is administered in the proper doses at the appropriate times. Improper administration of glycerol, which is an osmolite, may damage the RBCs. In particular, the RBCs may suffer osmolarity shock causing cellular damage if the glycerol is introduced too quickly.
The prior art method for washing the thawed, cryopreserved RBCs prior to transfusion is similarly time-consuming and labor intensive. More specifically, as described in the ""924 patent, the bag of frozen, cryopreserved RBCs is placed in a heated bath for 20-25 minutes to thaw the RBCs. A machine, such as the Model 115 from Haemonetics Corp., which includes a shaker platform, a centrifuge drive unit and a wash bowl is then employed. First, 50 ml of a 12% sodium chloride solution is gravity fed into the bag and mixed with the thawed RBCs by agitation of the shaker. The shaker is then turned off for two minutes to equilibrate the two solutions. Then shaker is then turned back on and 100 ml of a 0.9% sodium chloride and 0.2% glucose solution is gravity fed into the bag and mixed therein. Again, the shaker is stopped for about two minutes to permit the solutions to equilibrate. The shaker is turned on once again and 150 ml of the sodium chloride/glucose solution is added. The shaker is then turned off for two minutes. As with the glycerol solution, the introduction of each volume of wash solution to the thawed RBCs is also a delicate process. In particular, the wash solution is also an osmolite and thus may cause the RBC membranes to burst if it is added too quickly.
The contents of the bag are then centrifuged to remove the wash solution and removed glycerol and the resulting RBCs are transferred to a collection bag. Just prior to transfusion, the washed RBCs are concentrated through centrifugation and removal of the supernatant. The final RBCs typically have a hematocrit below 40%.
As the ""924 patent illustrates, the sensitivity of RBCs to osmolarity shock mandates repetitive, detailed steps that must be manually performed in order to avoid sudden changes in osmolite concentrationsxe2x80x94both when preparing the RBCs for freezing and also when recovering stored RBCs prior to use. This results in a costly process limiting the use of cryopreservation and thereby placing greater demands on timely blood collection efforts. In addition, due to chemical and other concerns, commercially available glycerol solutions typically come in rubber-sealed, glass bottles, rather than plastic bags having tube connections. To access the glycerol, a needle or spike connection must be used, thereby creating an xe2x80x9copenxe2x80x9d system which means potentially contaminated air and other impurities may enter the system. Furthermore, due to its high viscosity, glycerol cannot be gravity fed through an anti-bacterial filter. Thus, any contaminants entering the system are likely to reach the RBCs. Although this does not limit the length of time that the RBCs may remain frozen, it does require that the subsequently thawed red blood cells be utilized within 24 hours or discarded.
It is an object of the present invention to provide an improved method for preparing red blood cells for freezing.
It is a further object of the present invention to provide an improved method for rapidly delivering a cryopreservation solution to red blood cells without causing osmolarity shock.
It is a further object of the present invention to provide an improved method for rapidly washing thawed red blood cells without causing osmolarity shock.
Briefly, the invention relates to a system for delivering a cryopreservation solution to red blood cells to permit long-term, frozen storage and for subsequently removing the cryopreservative during red blood cell recovery. The system preferably includes a controller that is operably connected to a shaker platform, a centrifuge drive unit and one or more variable-speed pumps. A display screen, printer and input device may also be connected to the controller. A cryopreservation harness is used to connect a cryopreservation solution to a unit of concentrated red blood cells, which may be placed on the shaker platform. The controller is configured to monitor and govern the delivery of cryopreservation to the red blood cells via the pump. In particular, the controller periodically determines the amount of cryopreservation solution already added to the red blood cells and, based on this value, calculates a new cryopreservation solution flow rate in accordance with a novel algorithm. The algorithm provides a linear increase in red blood cell osmolarity that is selected to reduce the risk of shock and to minimize the processing time. By dynamically adjusting the pump speed, the controller delivers cryopreservation solution at the calculated flow rates throughout the cryopreservation process.
During the recovery process, a recovery harness, which includes a separation bowl and a wash solution, is similarly loaded onto the system. The thawed red blood cells are preferably placed on the shaker platform and coupled to both the wash solution and the separation bowl by two pumps. For red blood cell recovery, the controller is configured to monitor and govern the delivery of a first volume of wash solution to dilute the red blood cells. In particular, the controller dynamically adjusts the rate at which a dilution volume of wash solution is delivered to the red blood cells pursuant to a second novel algorithm. The second algorithm, which is similarly dependent on a calculated volume of wash solution already delivered, provides a linear decrease in red blood cell osmolarity that is selected to minimize shock while reducing the dilution time. By dynamically adjusting the speeds of the two pumps, the controller delivers wash solution at the calculated flow rates throughout the dilution process. After the red blood cells have been diluted, they may be transferred to the separation bowl and additional wash solution may be introduced in order to wash any remaining cryopreservative from the cells.
In the preferred embodiment, the cryopreservation solution is preferably pumped through an anti-bacterial filter before reaching the red blood cells. By filtering the cryopreservation solution and thereby removing potential contaminants, a significantly longer shelf-life of the subsequently recovered red blood cells is enabled. In addition, during the recovery process, the thawed red blood cells are initially diluted with a volume of a hypertonic solution prior the introduction of the wash solution. The hypertonic solution has an osmolarity close to that of the thawed red blood cells and further reduces the risk of shock. Furthermore, additional wash cycles are preferably performed to remove the debris resulting from the disintegration of weak cells unable to survive the recovery process and the washed red blood cells are suspended in a preservation solution to extend their shelf-life even further. Each step of the cryopreservation and recovery processes, moreover, is automatically performed by the system under the management of the controller, significantly reducing error and the overall processing time.